Assessing population-level variation in the mitochondrial genome of <Emphasis Type="Italic">Euphausia superba</Emphasis> using 454 next-generation sequencing

The Antarctic krill (Euphausia superba Dana 1852) is widely distributed throughout the Southern Ocean, where it provides a key link between primary producers and upper trophic levels and supports a major commercial fishery. Despite its ecological and commercial importance, genetic population structure of the Antarctic krill remains poorly described. In an attempt to illuminate genetic markers for future population and phylogenetic analysis, five E. superba mitogenomes, from samples collected west of the Antarctic Peninsula, were sequenced using new 454 next-generation sequencing techniques. The sequences, of lengths between 13,310 and 13,326 base pairs, were then analyzed in the context of two previously-published near-complete sequences. Sequences revealed relatively well-conserved partial mitochondrial genomes which included complete sequences for 11 of 13 protein-coding genes, 16 of 23 tRNAs, and the large ribosomal subunit. Partial sequences were also recovered for cox1 and the small ribosomal subunit. Sequence analysis suggested that the cox2, nad5, and nad6 genes would be the best candidates for future population genetics analyses, due to their high number of variable sites. Future work to reveal the noncoding control region remains.     

Draft genome sequence of Vibrio fischeri SR5, a strain isolated from the light organ of the Mediterranean squid Sepiola robusta

Here, we describe the draft genome sequence of Vibrio fischeri SR5, a squid symbiotic isolate from Sepiola robusta in the Mediterranean Sea. This 4.3-Mbp genome sequence represents the first V. fischeri genome from an S. robusta symbiont and the first from outside the Pacific Ocean.     

Mitochondrial DNA data indicate an introduction through Mainland Southeast Asia for Australian dingoes and Polynesian domestic dogs

In the late stages of the global dispersal of dogs, dingoes appear in the Australian archaeological record 3500 years BP, and dogs were one of three domesticates brought with the colonization of Polynesia, but the introduction routes to this region remain unknown. This also relates to questions about human history, such as to what extent the Polynesian culture was introduced with the Austronesian expansion from Taiwan or adopted en route, and whether pre-Neolithic Australia was culturally influenced by the surrounding Neolithic world. We investigate these questions by mapping the distribution of the mtDNA founder haplotypes for dingoes (A29) and ancient Polynesian dogs (Arc1 and Arc2) in samples across Southern East Asia (n = 424) and Island Southeast Asia (n = 219). All three haplotypes were found in South China, Mainland Southeast Asia and Indonesia but absent in Taiwan and the Philippines, and the mtDNA diversity among dingoes indicates an introduction to Australia 4600-18 300 years BP. These results suggest that Australian dingoes and Polynesian dogs originate from dogs introduced to Indonesia via Mainland Southeast Asia before the Neolithic, and not from Taiwan together with the Austronesian expansion. This underscores the complex origins of Polynesian culture and the isolation from Neolithic influence of the pre-Neolithic Australian culture.     

Limited effect of anti-rheumatic treatment on 15-prostaglandin dehydrogenase in rheumatoid arthritis synovial tissue

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E₂ (PGE₂) displays an important pathogenic role. The enzymes involved in its synthesis are highly expressed in the inflamed synovium, while little is known about 15- prostaglandin dehydrogenase (15-PGDH) that metabolizes PGE₂. Here we aimed to evaluate the localization of 15-PGDH in the synovial tissue of healthy individuals or patients with inflammatory arthritis and determine the influence of common RA therapy on its expression.

Methods

Synovial tissue specimens from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients were immunohistochemically stained to describe the expression pattern of 15-PGDH. In addition, the degree of enzyme staining was evaluated by computer analysis on stained synovial biopsies from two groups of RA patients, before and after RA specific treatment with either intra-articular glucocorticoids or oral methotrexate therapy. Prostaglandins derived from the cyclooxygenase (COX) pathway were determined by liquid-chromatography mass spectrometry in supernatants from interleukin (IL) 1β-activated fibroblast-like synoviocytes (FLS) treated with methotrexate.

Results

15-PGDH was present in healthy and inflamed synovial tissue, mainly in lining macrophages, fibroblasts and vessels. Intra-articular glucocorticoids showed a trend towards reduced 15-PGDH expression in RA synovium (p = 0.08) while methotrexate treatment left the PGE₂ pathway unaltered both in biopsies ex vivo and in cultured FLS.

Conclusions

Early methotrexate therapy has little influence on the expression of 15-PGDH and on any of the PGE₂ synthesizing enzymes or COX-derived metabolites. Thus therapeutic strategies involving blocking induced PGE₂ synthesis may find a rationale in additionally reducing local inflammatory mediators.     

Prime-boost vaccination with rBCG/rAd35 enhances CD8⁺ cytolytic T-cell responses in lesions from Mycobacterium tuberculosis-infected primates

To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I–restricted CD8+ cytolytic T cells using a recombinant Bacille Calmette-Guérin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8⁺ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3⁺ and CD8⁺ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8⁺ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8+ effector T-cell responses at the local site of infection in Mtb-challenged animals.     

Deimatic Display in the European Swallowtail Butterfly as a Secondary Defence against Attacks from Great Tits

Background

Many animals reduce the risk of being attacked by a predator through crypsis, masquerade or, alternatively, by advertising unprofitability by means of aposematic signalling. Behavioural attributes in prey employed after discovery, however, signify the importance of also having an effective secondary defence if a predator uncovers, or is immune to, the prey’s primary defence. In butterflies, as in most animals, secondary defence generally consists of escape flights. However, some butterfly species have evolved other means of secondary defence such as deimatic displays/startle displays. The European swallowtail, Papilio machaon, employs what appears to be a startle display by exposing its brightly coloured dorsal wing surface upon disturbance and, if the disturbance continues, by intermittently protracting and relaxing its wing muscles generating a jerky motion of the wings. This display appears directed towards predators but whether it is effective in intimidating predators so that they refrain from attacks has never been tested experimentally.

Methodology/Principal Findings

In this study we staged encounters between a passerine predator, the great tit, Parus major, and live and dead swallowtail butterflies in a two-choice experiment. Results showed that the dead butterfly was virtually always attacked before the live butterfly, and that it took four times longer before a bird attacked the live butterfly. When the live butterfly was approached by a bird this generally elicited the butterfly’s startle display, which usually caused the approaching bird to flee. We also performed a palatability test of the butterflies and results show that the great tits seemed to find them palatable.

Conclusions/Significance

We conclude that the swallowtail’s startle display of conspicuous coloration and jerky movements is an efficient secondary defence against small passerines. We also discuss under what conditions predator-prey systems are likely to aid the evolution of deimatic behaviours in harmless and palatable prey.     

Meta-analysis of genome-wide scans for total body BMD in children and adults reveals allelic heterogeneity and age-specific effects at the WNT16 locus

To identify genetic loci influencing bone accrual, we performed a genome-wide association scan for total-body bone mineral density (TB-BMD) variation in 2,660 children of different ethnicities. We discovered variants in 7q31.31 associated with BMD measurements, with the lowest P = 4.1 × 10(-11) observed for rs917727 with minor allele frequency of 0.37. We sought replication for all SNPs located ± 500 kb from rs917727 in 11,052 additional individuals from five independent studies including children and adults, together with de novo genotyping of rs3801387 (in perfect linkage disequilibrium (LD) with rs917727) in 1,014 mothers of children from the discovery cohort. The top signal mapping in the surroundings of WNT16 was replicated across studies with a meta-analysis P = 2.6 × 10(-31) and an effect size explaining between 0.6%-1.8% of TB-BMD variance. Conditional analyses on this signal revealed a secondary signal for total body BMD (P = 1.42 × 10(-10)) for rs4609139 and mapping to C7orf58. We also examined the genomic region for association with skull BMD to test if the associations were independent of skeletal loading. We identified two signals influencing skull BMD variation, including rs917727 (P = 1.9 × 10(-16)) and rs7801723 (P = 8.9 × 10(-28)), also mapping to C7orf58 (r(2) = 0.50 with rs4609139). Wnt16 knockout (KO) mice with reduced total body BMD and gene expression profiles in human bone biopsies support a role of C7orf58 and WNT16 on the BMD phenotypes observed at the human population level. In summary, we detected two independent signals influencing total body and skull BMD variation in children and adults, thus demonstrating the presence of allelic heterogeneity at the WNT16 locus. One of the skull BMD signals mapping to C7orf58 is mostly driven by children, suggesting temporal determination on peak bone mass acquisition. Our life-course approach postulates that these genetic effects influencing peak bone mass accrual may impact the risk of osteoporosis later in life.     

Human IgG/FcγR Interactions Are Modulated by Streptococcal IgG Glycan Hydrolysis

Background

The human pathogen Streptococcus pyogenes produces an endoglycosidase, EndoS that hydrolyzes the chitobiose core of the asparagine-linked glycan on the heavy chain of human IgG. IgG-binding to Fc gamma receptors (FcγR) on leukocytes triggers effector functions including phagocytosis, oxidative burst and the release of inflammatory mediators. The interactions between FcγR and the Fc domain of IgG depend on the IgG glycosylation state.

Methodology/Principal Findings

Here we show for the first time that EndoS hydrolyzes the heavy chain glycan of all four human IgG subclasses (IgG1-4), in purified form and in a plasma environment. An inactive form of EndoS, obtained by site-directed mutagenesis, binds IgG with high affinity, in contrast to wild type EndoS that only transiently interacts with IgG, as shown by Slot-blotting and surface plasmon resonance technology. Furthermore, EndoS hydrolysis of the IgG glycan influences the binding of IgG to immobilized soluble FcγR and to an erythroleukemic cell line, K562, expressing FcγRIIa. Incubation of whole blood with EndoS results in a dramatic decrease of IgG binding to activated monocytes as analyzed by flow cytometry. Moreover, the IgG bound to K562 cells dissociates when cells are treated with EndoS. Likewise, IgG bound to immobilized FcγRIIa and subsequently treated with EndoS, dissociates from the receptor as analyzed by surface plasmon resonance and Western blot.

Conclusions/Significance

We provide novel information about bacterial enzymatic modulation of the IgG/FcγR interaction that emphasizes the importance of glycosylation for antibody effector functions. Moreover, EndoS could be used as a biochemical tool for specific IgG N-glycan hydrolysis and IgG purification/detection, or as a potential immunosuppressing agent for treatment of antibody-mediated pathological processes.     

Stimulated efflux of amino acids and glutathione from cultured hippocampal slices by omission of extracellular calcium: likely involvement of connexin hemichannels

Omission of extracellular Ca(2+) for 15 min from the incubation medium of cultured hippocampal slices stimulated the efflux of glutathione, phosphoethanolamine, hypotaurine, and taurine. The efflux was reduced by several blockers of gap junctions, i.e. carbenoxolone, flufenamic acid, and endothelin-1, and by the connexin43 hemichannel blocking peptide Gap26 but was unchanged by the P2X(7) receptor inhibitor oxidized ATP, a pannexin1 hemichannel blocking peptide and an inactive analogue of carbenoxolone. Pretreatment of the slices with the neurotoxin N-methyl-d -aspartate left the efflux by Ca(2+) omission unchanged, indicating that the stimulated efflux primarily originated from glia. Elevated glutamate efflux was detected when Ca(2+) omission was combined with the glutamate uptake blocker l-trans-pyrrolidine-2,4-dicarboxylate and when both Ca(2+) and Mg(2+) were omitted from the medium. Omission of Ca(2+) for 15 min alone did not induce delayed toxicity, but in combination with blocked glutamate uptake, significant cell death was observed 24 h later. Our results indicate that omission of extracellular Ca(2+) stimulates efflux of glutathione and specific amino acids including glutamate via opening of glial hemichannels. This type of efflux may have protective functions via glutathione efflux but can aggravate toxicity in situations when glutamate reuptake is impaired, such as following a stroke.